Project Summary/Abstract Fluorescence has significant potential for biomedical imaging applications because of the relatively low cost of imaging equipment, the nominal toxicity of non-ionizing radiation (i.e., light), the potential for molecular imaging using target-specific contrast agents, and the prospect of multiplexed imaging using discretely colored fluorophores. Molecules common in biological tissues including lipids, water, and hemoglobin scatter and absorb light, rendering tissue opaque to visible wavelengths, but longer, near infrared (NIR) wavelengths penetrate deeper, giving us an optical window into the body. To see inside a tissue, we require bright, photostable, highly absorbing, NIR fluorophores. Despite exceptional results in vitro, we can improve on the in vivo performance of organic dyes, fluorescent proteins, and traditional semiconductor quantum dots (QDs), which are typically dim, toxic, not red enough, or all of the above. We have created a material that literally flips a quantum dot inside out to make a quantum shell (QS) comprised of non-toxic elements (In, P, Se, Zn, S) that is tunable from 500 ? 900 nm. Because InP absorbs more efficiently than CdSe, these materials are brighter than previous materials with a smaller size, while emitting in the NIR and reducing toxicity. We propose a technology development plan that would enable us to refine the structural and optical properties of these particles to generate a brightness-matched palette of fluorophores to enable multiplexing in deep tissue. We will deploy these particles in widefield imaging and multiphoton microscopy (MPM) experiments to first objectively quantify and then demonstrate the optical superiority of these probes. After evaluating the in vitro and in vivo biocompatibility of various formulations of water-soluble QSs, we will use targeted and untargeted QSs together for dual probe imaging of cell surface biomarkers to selectively highlight a xenograft tumor. In addition to widefield imaging, we will objectively evaluate the MPM contrast of the QSs. The exceptionally high absorptivity of the particles ensures high two- and three- photon action cross-sections. We will quantitatively compare the brightness and tissue penetration depth of the InP QSs against other red and NIR fluorophores. Synthetic iterations to the particles will use the unique particle geometry to generate QSs with varying emission colors, but the same brightness. We will compare zwitterionic coatings to our benchmark lipid-PEG coating to try to enhance imaging contrast through longer circulation time and more efficient targeting. The success of this project will yield a rainbow of non-toxic, NIR fluorophores that can be used collectively could transform preclinical molecular imaging.